The goal of our grant is to understand the signaling steps linking the endoplasmic reticulum (ER) localized ER Ca2+ sensors STIM1 and STIM2 to the activation of Ca2+ influx across the plasma membrane (PM): store- operated Ca2+-influx (SOC). To gain insight into cellular functions of STIM proteins, we will determine their contribution to regulating the collective migration of endothelial cells and their role at ER-PM junctions in regulating the rate of transfer of hydrophobic molecules between the ER and PM. This work is significant due to an important role of the STIM signaling pathway in different immune and cardiac diseases. Our study builds on a human siRNA screen that we performed during the last funding period in which we discovered STIM proteins as an essential component of SOC. We characterized both STIM1 and STIM2 proteins and showed that they function as the elusive Ca2+ sensors that monitor the Ca2+ level in the lumen of the ER and that a constitutively active version is sufficient to activate PM Ca2+ influx. We further showed that STIM proteins rapidly oligomerize following luminal Ca2+ dissociation (upon receptor triggered IP3-mediated Ca2+ release), and translocate within the ER membrane to a newly characterized site in the PM, the ER-PM junction. At these sites, STIM interacts with PM Ca2+ channels Orai1-3 and other putative targets. While much has been learned about the activation and function of STIM proteins, many questions remain unresolved. We will make use of live-cell imaging, site-directed mutagenesis, biochemical assays and modeling approaches to answer three important outstanding questions about STIM-mediated signal transduction and ER-PM junctions. We will first focus on establishing a molecular basis for this intriguing new inside-out signaling paradigm, by investigating the Ca2+ sensing, oligomerization and plasma membrane translocation mechanisms of STIM1 and STIM2. Second, we will focus on understanding the role of Ca2+ and STIM proteins in endothelial migration by building on our initial findings that migrating cells markedly polarize the distribution of STIM to the front, contributing to the generation of local Ca2+ pulses and local actin contraction. Finally, based on our initial evidence, we will focus on the role of STIM and ER-PM junctions in regulating the transfer of phosphatidylinositol lipids, cholesterol, and Ras proteins between the ER and PM. This is a new paradigm for why ER-PM junctions exist in cells and also implies a new role for STIM proteins.